The genetic code of most organisms was evolved to encode 20 amino acids. Although the ability to encode 20 amino acids provides the basis to translate proteins necessary for life, researchers are also limited to these 20 amino acids for conventional site-directed mutagenesis. The ability to encode unnatural amino acids provides researchers the ability to circumvent the limitation imposed by the genetic code. Genetically encoding unnatural amino acids provides researchers the means to not only mimic naturally occurring posttranslational modifications but also the ability to encode amino acids with new physical or chemical properties to study biological processes. The incorporation of unnatural amino acids into proteins had been developed in Escherichia coli and also in yeast. We have developed a methodology to genetically incorporate unnatural amino acids in mammalian cells in response to an amber codon (UAG). The incorporation of unnatural amino acids is high in E. coli and yeast, but the incorporation in mammalian cells is relatively low. In addition to developing the system to incorporate unnatural amino acids in mammalian cells, we have also improved suppression efficiencies by modifying the synthetase and unnatural amino acid. To incorporate unnatural amino acids in response to an amber codon, the tRNA anticodon is mutated from a GUA to a CUA. We were able to show that engineering the anticodon-binding domain of the synthetase could enhance the recognition of the tRNA and thus increased suppression efficiencies. Furthermore, by masking the carboxyl group of the amino acid by an ester group, we were able to increase the bioavailability of an unnatural amino acid to further increase suppression efficiencies. Most evolved synthetases aminoacylate unnatural amino acids that are structurally similar to the native substrate of the wild-type synthetase. We were able to adapt Methanosarcina mazei pyrrolysine synthetase (PylRS) to charge a considerable disparate amino acid from its native substrate, O-methyl-L-tyrosine. In addition, the X-ray crystal structure was solved for the evolved PylRS complexed with O-methyl-L-tyrosine at 1.75Å. This multifaceted approach provides the basis to engineer the PylRS to incorporate a significantly diverse selection of unnatural amino acids than previously anticipated.

By combining the tools of organic chemistry with those of physical biochemistry and cell biology, Non-Natural Amino Acids aims to provide fundamental insights into how proteins work within the context of complex biological systems of biomedical interest. The critically acclaimed laboratory standard for 40 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. With more than 400 volumes published, each Methods in Enzymology volume presents material that is relevant in today's labs -- truly an essential publication for researchers in all fields of life sciences. - Demonstrates how the tools and principles of chemistry combined with the molecules and processes of living cells can be combined to create molecules with new properties and functions found neither in nature nor in the test tube - Presents new insights into the molecular mechanisms of complex biological and chemical systems that can be gained by studying the structure and function of non-natural molecules - Provides a "one-stop shop" for tried and tested essential techniques, eliminating the need to wade through untested or unreliable methods

This detailed volume explores non-canonical amino acids (ncAAs) through their site-specific incorporation by genetic code expansion (GCE). The collection provides a broad resource of methods for implementing GCE in E. coli, mammalian cells, and animals, highlighting specific applications ranging from fluorescence labeling to photocontrol and the study of protein post-translational modification. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Genetically Incorporated Non-Canonical Amino Acids: Methods and Protocols serves as an ideal source of methodologies that can be adapted and extended, migrated to different model systems, and combined in new ways to help explore a wide range of biological questions and to augment industrial and pharmaceutical protein engineering.

Dear Colleagues, Synthetic biology is a broad and emerging discipline that capitalizes on recent advances in molecular biology, genetics, protein and RNA engineering and omics technologies. These technologies have transformed our ability to reveal the biology of the cell and the molecular basis of disease. This Special Issue on “Synthetic RNA and DNA Programming” features original research articles and reviews, highlighting novel aspects of basic molecular biology and the molecular mechanisms of disease that were uncovered by the application and development of novel synthetic biology-driven approaches.

The ability to introduce non-canonical amino acids in vivo has greatly expanded the repertoire of accessible proteins for basic research and biotechnological application. Here, the different methods and strategies to incorporate new or modified amino acids are explained in detail, including a lot of practical advice for first-time users of this powerful technique. Novel applications in protein biochemistry, genomics, biotechnology and biomedicine made possible by the expansion of the genetic code are discussed and numerous examples are given. Essential reading for all molecular life scientists who want to stay ahead in their research.

This detailed volume provides in-depth protocols for protein labeling techniques and applications, with an additional focus on general background information on the design and generation of the organic molecules used for the labeling step. Chapters provide protocols for labeling techniques and applications, with an additional focus on general background information on the design and generation of the organic molecules used for the labeling step. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Site-Specific Protein Labeling: Methods and Protocols provides a comprehensive overview on the most relevant and established labeling methodologies, and helps researchers to choose the most appropriate labeling method for their biological question.

A one-stop reference that reviews protein design strategies to applications in industrial and medical biotechnology Protein Engineering: Tools and Applications is a comprehensive resource that offers a systematic and comprehensive review of the most recent advances in the field, and contains detailed information on the methodologies and strategies behind these approaches. The authors—noted experts on the topic—explore the distinctive advantages and disadvantages of the presented methodologies and strategies in a targeted and focused manner that allows for the adaptation and implementation of the strategies for new applications. The book contains information on the directed evolution, rational design, and semi-rational design of proteins and offers a review of the most recent applications in industrial and medical biotechnology. This important book: Covers technologies and methodologies used in protein engineering Includes the strategies behind the approaches, designed to help with the adaptation and implementation of these strategies for new applications Offers a comprehensive and thorough treatment of protein engineering from primary strategies to applications in industrial and medical biotechnology Presents cutting edge advances in the continuously evolving field of protein engineering Written for students and professionals of bioengineering, biotechnology, biochemistry, Protein Engineering: Tools and Applications offers an essential resource to the design strategies in protein engineering and reviews recent applications.

The genetic code was deciphered experimentally around 1966 and for a number of years scientists considered it to be "universal" for all forms of life. In 1981 researchers shocked the scientific community with the discovery that the code differed in mitochondria and certain other organisms, evidence that the genetic code was still evolving. This book discusses the distribution and origin of the non-universal codes and examines the possible mechanisms of code changes, making it essential reading for all those interested in evolutionary genetics.

This book addresses the design of emerging conceptual tools, technologies and systems including novel synthetic parts, devices, circuits, oscillators, biological gates, and small regulatory RNAs (riboregulators and riboswitches), which serve as versatile control elements for regulating gene expression. Synthetic biology, a rapidly growing field that involves the application of engineering principles in biology, is now being used to develop novel systems for a wide range of applications including diagnostics, cell reprogramming, therapeutics, enzymes, vaccines, biomaterials, biofuels, fine chemicals and many more. The book subsequently summarizes recent developments in technologies for assembling synthetic genomes, minimal genomes, synthetic biology toolboxes, CRISPR-Cas systems, cell-free protein synthesis systems and microfluidics. Accordingly, it offers a valuable resource not only for beginners in synthetic biology, but also for researchers, students, scientists, clinicians, stakeholders and policymakers interested in the potential held by synthetic biology.